In vitro and in vivo ligand binding to the 5HT3 serotonin receptor characterised by time-resolved fluorescence spectroscopy

The binding of the fluorescein-labeled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino- (N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilized ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT3R), was characterized by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was obsd. for the GR-flu - receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by detg. the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation expts. indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, the authors succeeded in measuring the binding of GR-flu to 5HT3 receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomol. interactions in live cells. [on SciFinder (R)]

Ligand binding to the serotonin 5HT3 receptor studied with a novel fluorescent ligand

Alain Bernard, Rudolf Hovius, Horst Pick, Horst Vogel

The thermodn. and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluorescein-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiol. investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissocn. const. of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissocn. consts. of .apprx.0.2 nM. Fluorescence measurements showed that selective 5HT3 receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [3H]-GR65630. The kinetics of GR-flu binding to the 5HT3 receptor revealed a bimol. assocn. process with an on-rate const. of 1.17106 s-1 M-1 and a biphasic dissocn. reaction with off-rate consts. of 27510-6 and 43*10-6 s-1. The temp. dependence of the dissocn. const. yielded an enthalpic term of -26 kJ mol-1 and an entropic term of 94 J K-1 mol-1 for the binding of GR-flu to the receptor, indicating that both quantities contribute equally to the reaction. An activation enthalpy DH#on and entropy DS#on of binding of 50 kJ mol-1 and 43 J mol-1 K-1 were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT3 receptor. The fluorescence anisotropy of receptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it is 0.8 pH unit more acidic than the bulk soln. [on SciFinder (R)]

1998

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: From biosynthesis to endocytosis

Cédric Deluz, Erwin Ilegems, Horst Pick, Horst Vogel

Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chem. blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quant. to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was obsd. within minutes after exposure to an extracellular agonist. The authors' orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking. [on SciFinder (R)]

2004

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

The thesis presented here describes studies of ligand binding, function, and arrangement of ligand-gated ion channels in cell membranes. Fluorescence microscopy and the patch clamp technique are used to investigate the nicotinic acetylcholine receptor and the serotonergic 5-HT3 receptor. Charge effects on ligand binding were investigated on the 5-HT3 receptor by site directed mutagenesis of strongly conserved charged amino acids close to the ligand-binding site. It turns out that steric effects have a higher impact on ligand binding than charges but that charged amino acids change the local environment strongly. This was exploited using a fluorescently labelled ligand to determine the distance of the bound ligand to the mutated amino acid. Ligand binding to the acetylcholine receptor was studied with a novel fluorescent ligand based on a toxin from the venom of marine snails (α-conotoxin GI).The fluorescent toxin acts as antagonist and can be bind completely reversible and with excellent specificity to muscle type acetylcholine receptors. Repetitive cycles of binding and unbinding were performed to obtain binding kinetics on single cells.The developed fluorescence microscope experiment enables the detection of only very low amount of fluorescent ligand from around 103 down to single molecules. The reversible binding was exploited to label single acetylcholine receptor and to monitor their diffusion in cell membranes. HEK293 cells served as a model system where co-expression of the anchoring protein rapsyn strongly influences receptor diffusion. Data were compared to diffusion in muscle cell lines during differentiation into myotubes. To obtain new information on ligand binding and channel gating, an experiment has been developed to simultaneously measure fluorescence and ion channel activity. The fluorescently labelled α-conotoxin GI was used to demonstrate the capability of this system. In addition, to increase the yield of electrophysiological measurements, a technique to perform patch clamp on a chip was further developed towards automation.

EPFL2005

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

EPFL2005